Paper disk

S. aureus CCARM 3A048 and E. faecium CCARM 5202 were grown at 37 °C for 24 h in BHI broth medium (calf brains 7.7 g, beef heart 9.8 g, protease peptone 10.0 g, dextrose 2.0 g, sodium chloride 5.0 g, disodium phosphate 2.5 g/L, pH 7.2–7.4) with shaking at 150 rpm. Cultures (0.1 mL) were then spread on BHI agar plates and paper disks (8 mm, Advantec, Toyo Roshi Kaisha. Ltd., Tokyo, Japan) sterilized with UV for 10 min, spotted with extracts (0.1 mL), and dried for 2–3 h, were placed on plates with spotted surfaces contacting the agar. For primary screening, aliquots of five extracts (1.0 mg each) were placed on a plate. Control of 10% EA or 10% DMSO, and vancomycin (20 or 40 ppm for E. faecium CCARM 5202 and S. aureus CCARM 3A048, respectively) were included. Plates were incubated at 37 °C for 12 h and the diameters of halos around the disks were observed or measured. Effective samples were selected by second screening (conducted in duplicate) using 0.5 mg of extract.

To determine the antimicrobial activity of LAB cultured in MRS or juice, spot‐on‐the‐lawn assays (Lee & Chang, 2016) or paper disk assays (Yang & Chang, 2010) were used. Briefly, LAB strain was cultivated in MRS broth at 30℃ for 24 hr or in juice at 15℃ for 0–5 days. The culture was then centrifuged (10,100 g, 15 min, 4℃) and the supernatant sterilized by passing through a 0.45 μm membrane filter (Sartorius) and used as culture filtrate or juice filtrate.
The prepared culture filtrate or juice filtrate was used for the antimicrobial activity assay. Plates were prepared by adding fungi (6.0 log spore per 20 ml MEA or PDA) to 1.5% bacto agar (Duchefa) for antifungal assays or by spreading the bacteria (6.0 log CFU/mL) onto MRS, LB, TSB, or NB + 2% NaCl agar for antibacterial assays (Lee & Chang, 2016; Yang & Chang, 2010). For the paper disk assay, paper disks (diameter 8 mm; Advantec, Tokyo, Japan) on MRS plates were spotted with 100 µl of sample. The plates were then incubated at 30℃ for 24 hr and examined for inhibition zones. For the spot‐on‐the‐lawn assay, 10 µl of sample was spotted onto the sensitive mold and bacterial plates. Antimicrobial activity, which was defined as the reciprocal of the highest dilution at which microbial growth was inhibited, was expressed as arbitrary units (AU) per milliliter.

Decayed stakes were dried using a freeze-drier and ground with a mortar and pestle. Five grams of the ground stake was extracted using 100 mL n-hexane at room temperature for 24 h. The extract was concentrated to 10 mL in a rotary evaporator at 10 °C. The concentration of the extract obtained by this procedure was 8%. The n-hexane extract of a sound stake was prepared in the same manner.
Paper disks (8 mm in diameter, thick type; ADVANTEC TOYO, Tokyo, Japan) were treated with the concentrated extract and kept at room temperature to remove the solvent. No-choice feeding tests were conducted using disks following the method described in previous section, with the exception that 50 workers were introduced per cup. This test was conducted using a single termite colony originating from the RISH field. Paper disks immersed in the solvent alone served as controls. Five replicates were tested for 10 days at 27 °C. After the test, the Paper disks were removed, cleared of any attached debris, oven-dried at 60 °C for 48 h, and weighed. The mass loss of the disks was calculated from the difference in dry weight before and after the exposure.

Paper disks (8 mm in diameter, thick type; ADVANTEC TOYO, Tokyo, Japan) were treated with the extract obtained from laboratory-decayed wood. A sound wood extract (8%), was prepared under conditions similar to the decayed wood extract. This experiment was conducted to validate that feeding deterrence was not correlated with the sound wood extract. Extract-treated Paper disks were kept at room temperature to remove the solvent used in the extraction process. No-choice feeding tests were conducted with the Paper disks using 50 termite workers that were introduced into each cup. A single colony from Oarai City, Ibaraki Prefecture, was used in this test. Paper disks immersed in the solvent served as controls. Three replicates were tested for 10 days at 27 °C. After the test, the Paper disks were removed, cleared of any attached debris, oven-dried at 60 °C for 48 h, and weighed. The mass losses of the disks were calculated from the difference in dry weight before and after exposure.